Protective Effect Of Targeting ER Stress And HO-1 On Ischemia Reperfusion Injury In Vascularized Composite Allotransplantation

BackgroundThe field of vascularized composite allotransplantation(VCA)is developing,but the shortage of donors,and acute and chronic graft rejection are the limitations of VCA.Ischemia-reperfusion injury(IRI)is inevitable in the process of graft acquisition,storage,and transport.The use of donors who sustained longer ischemic time could alleviate the problem of donor shortage.However,numerous studies have demonstrated that prolonged ischemic time of organ and graft is associated with tissue damage,and acute and chronic rejection episodes.Therefore,minimizing IRI can improve organ utilization and reduce the incidence of VCA rejection.The mechanism and therapeutic experience of VCA IRI mainly referred to solid organ transplantation and limb replantation.There is still a lack of specific clinical research and animal experiments to clarify the critical ischemic time of VCA.Despite exploring the critical ischemic time in different tissues of VCA,analyzing the trend of protein change after reperfusion injury may help us to reveal the underlying mechanism of VCA IRI,which can lay a foundation for the subsequent targeted therapy.Endoplasmic reticulum stress(ER stress)and heme oxygenase-1(HO-1)signaling pathways have been confirmed to be involved in organ reperfusion injury.Elimination of ER stress and HO-1 induction can alleviate tissue damage and inflammatory infiltration after organ reperfusion.Thus,targeting ER stress and HO-1 may alleviate VCA tissue damage.To further verify the above hypothesis,targeting ER stress by 4-phenyl butyric acid(4-PBA),and HO-1 induction by cobalt protoporphyrin(Co PP),or Co PP-modified macrophage therapy(Co PP-BMDM)were used to explore its mechanism in VCA IRI.Chapter 1 Evaluation of the critical ischemic time of VCAObjective: To evaluate the critical ischemic time of VCA graft.Materials and methods: Thirty BALB/c male mice were purchased as donors,and 30 C57BL/6 male mice were purchased as recipients.A mouse model of IRI with allogeneic hindlimb transplantation was established.Group 1: donor cold ischemia time 6h;Group 2: donor cold ischemia time 12h;Group 3: donor cold ischemia time 24 h.The transplanted limbs were successfully vascularized and perfused for 24 hours,and skin and muscle tissue samples were collected.HE staining was used to evaluate the early VCA tissue damage.Then,the hindlimb transplant was continuously monitored for 7 days.The circumference of donor hindlimb and the survival status of recipients were recorded.Results: The survival rate was only 80% when the cold ischemia time was prolonged to 24 h.Histological staining showed that the skin was not sensitive to ischemia.Muscle tissue is sensitive to ischemia,tissue damage and the score of inflammatory infiltration are proportional to cold ischemia time.Conclusion: The critical ischemia time of VCA graft is 24 h.Chapter 2 The protein changing trends of VCA after IRIObjective: To analyze the protein trends of VCA after IRI and to lay a foundation for further exploration of its molecular mechanism.Materials and methods: Thirty BALB/c male mice were purchased as donors,and 30 C57BL/6 male mice were purchased as recipients.Five C57BL/6 male mice were purchased as a sham-operated group.All the donor hindlimb cold ischemia time was 24 h.Skin and muscle tissue samples were collected at POD1,POD3,POD5,POD7,POD14 and POD28,histological staining and Westen Blot were used to analyze these samples.Results: In skin and muscle,the changes of HO-1 protein showed the same trend.The expression of HO-1 increased on POD 1,reached to peak on POD 5,and then gradually decreased.The changes of CHOP protein in skin and muscle also showed the same trend,reaching the peak on POD 1and POD5 respectively and then decreased and remained stable after PODConclusion: CHOP and HO-1 proteins are significantly increased in the early stage of IRI,targeting endoplasmic reticulum stress and HO-1may alleviate tissue damage.Chapter 3 The mechanism of targeting ER stress attenuates VCA IRIObjective: To Explore the mechanism of targeting ER stress in alleviation of IRI.Materials and methods: Thirty-five BALB/c male mice were purchased as donors,and 35 C57BL/6 male mice were purchased as recipients.Ten C57BL/6 male mice were purchased as a sham-operated group.All the donor hindlimb cold ischemia time was 24 h.Group 1: 24 h reperfusion+PBS control;Group 2: 24 h reperfusion+4-PBA(200mg/kg/day);Group 3: 24 h reperfusion+4-PBA(300mg/kg/day);Group 4: 24 h reperfusion+4-PBA(450mg/kg/day);Group 5: 72 h reperfusion+PBS control;Group 6: 72 h reperfusion+4-PBA(100mg/kg/day);Group 7: 72 h reperfusion+4-PBA(200mg/kg/day);In all transplanted groups,the 24 h reperfusion group was not given rapamycin,while the 72 h reperfusion group was given rapamycin(5mg/kg/day)for anti-rejection purposes.Skin and muscle samples were collected at 24 h or 72 h after reperfusion,histological staining was used to assess tissue necrosis and inflammatory infiltration.The expression levels of CHOP,DR-5,Cleaved caspase-3,LC3 B,and Vinculin in skin and muscle were detected by Western Blot.Results: When the dose of 4-PBA was increased to 450 mg/kg/day,the expression of autophagy protein LC3 B was significantly increased,and the expression of CHOP and DR5 protein was decreased(P<0.05).Histological staining showed that the proportion of muscle necrosis in the4-PBA treatment(450 mg/kg/day)was significantly lower than that in the PBS control group,and the score of inflammatory infiltration was lower compared with the control group.Conclusion: 4-PBA may alleviate muscle damage at 24 h reperfusion by enhancing autophagy and inhibiting ER stress-related apoptosis.Chapter 4 The protective effect of high expression HO-1 on VCA IRIObjective: To know the protective mechanism of high expression of HO-1 in VCA IRI.Materials and methods: Twenty BALB/c male mice were purchased as donors,and 20 C57BL/6 male mice were purchased as recipients.Ten C57BL/6 male mice were purchased,of which 5 mice were used as sham operation group,and the other 5 mice were used as bone marrow-derived macrophages(BMDM)donors.All the donor hindlimb cold ischemia time was 24 h.Group 1: PBS control;Group 2: Co PP treatment;Group 3:Control-BMDM treatment;Group 4: Co PP-BMDM treatment.Skin and muscle samples were collected at 24 h after reperfusion for histological staining to assess tissue necrosis and inflammatory infiltration.The expression levels of HO-1,p-STAT3,Cleaved caspase-3,β-Catenin,and Vinculin in skin and muscle were detected by Western Blot.Results: Compared with the PBS group,Co PP treatment significantly increased the expression of HO-1 in skin and muscle and decreased the expression of Cleaved Caspase-3 in muscle tissue on POD 1(P<0.05).Co PP treatment also reduced the proportion of muscle necrosis and inflammatory infiltration compared to PBS control.Compared with the Control-BMDM group,Co PP-BMDM treatment significantly increased the expression of HO-1 and decreased the expression of Cleaved Caspase-3 in skin and muscle(P<0.05).Histological staining showed that Co PPBMDM treatment reduced the proportion of muscle necrosis and inflammatory infiltration compared to Control-BMDM group.Conclusion: Both Co PP and Co PP-BMDM treatments significantly increased the expression levels of HO-1 in skin and muscle and alleviated IRI in VCA graft.